This invention is directed to a chromogenic compound, a thiophospholipid enzyme substrate, specifically thiophosphatidyl ethyleneglycol (thioPEG), which is useful as an indicator compound in an analytical test system. In particular, the present invention relates to a novel chromogenic substrate compound, its preparation and use in an assay and for the spectrophotometric detection of enzymes in a liquid test sample.
All biological membranes have the same classes of chemical compounds and a number of properties in common. These membranes are very dynamic structures with a movement that permits the cell as well as subcellular structures to adjust their shape and to move. The chemical components of membranes, which include lipids and protein, are well suited for the dynamic movement of membranes. Further, these membranes control the composition of the space they enclose by their ability to exclude a variety of molecules and via selective transport systems which permit the movement of molecules from one side to another. By controlling the translocation of substrates, cofactors and ions from one compartment to another, membranes modulate the concentration of substances, thereby exerting a strong influence on the body""s metabolic pathways.
Lipids are a major component of membranes. The three major lipid components of cell membranes are phosphoglycerides, sphingolipids, and cholesterol. The phosphoglycerides and sphingomyelin, a sphingolipid containing phosphate, are classified as phospholipids.
Phospholipids, which are waxy solids, are found almost exclusively in cellular membranes and in the lipoproteins of blood plasma. Phospholipids thus serve primarily as structural elements and are never stored in large amounts. As their name implies, this group of lipids contains phosphorus in the form of phosphoric acid. The major phospholipids found in cells contain two fatty acid molecules which are esterified to the first and second hydroxyl groups of glycerol. The third hydroxyl group, at carbon atom 3, is esterified with phosphoric acid. Phospholipids contain a second alcohol which is also esterified to the phosphoric acid to form a phosphodiester; the second alcohol group is thus located on the polar head of the phospholipid molecule. The general structural formula of the phospholipids is shown below. ROxe2x80x94denotes the second alcohol group. 
Different types of phospholipids are named according to the second alcohol at their polar heads. The most abundant phospholipids are the closely related phosphatidylethanolamine (also called cephalin) and phosphatidylcholine (also called lecithin), which contain ethanolamine and choline, respectively, at their heads. Each of these can occur in different forms depending on the fatty acids they contain.
Phospholipids readily undergo hydrolysis, catalyzed by acids, bases, or enzymes. Dilute base removes the two fatty acid groups of phosphatidylcholine, leaving the rest of the molecule intact. Strong base causes cleavage of both the fatty acids as well as the choline, leaving glycerol 3-phosphate, which can then be cleaved to yield glycerol and phosphoric acid by boiling with hydrochloric acid.
Different types of phospholipases are categorized based on the specific linkage for which they catalyze hydrolysis in the phospholipid molecule. Sites of action of phospholipases A1, A2, C and D on phosphatidylcholine are shown below. 
Via assay, phospholipase enzymatic activity may be measured.
The determination of phospholipase enzymes via assay is important in a variety of fields such as biochemical research, environmental and industrial testing, and medical diagnostics. The quantitation of enzyme levels in body fluids such as serum and plasma provides very useful information to the physician in diagnosing disease states and their treatment. In addition to being analytes of interest in biological fluids, enzymes can also serve as detection reagents in a variety of analytical systems such as immunoassays and nucleic acid hybridization techniques. In such systems, enzymes are useful directly or indirectly as indicators to monitor the extent of antigen-antibody binding or nucleic acid hydridization that occurs.
Accordingly, the desire to detect enzyme analyte and to use enzyme labels as a diagnostic tool in various analytical test systems has given rise to the development of optical indicator compounds for use in the detection and measurement of the activity of such enzymes. Typically, such known optical indicator compounds comprise a detectable chemical group, such as a fluorogen or a chromogen, which has been derivatized with an enzyme cleavable substrate group specific for the enzyme of interest. Such optical indicator. compounds exhibit an optical signal which is different from the optical signal which is provided by the cleaved native form of the fluorogen or chromogen. In principle, the enzyme cleaves the indicator compound to liberate the chromogen in the form of a distinctly fluorescent or colored product to provide a change in fluoroescence or color which is proportional to the amount of enzyme present which, in turn, can be correlated to the amount of analyte present in a liquid test sample.
Currently, there are numerous methods used to measure phospholipase enzymatic activity in an assay. Phospholipases are capable of cleaving thio ester bonds of an unnatural substrate. For example, phospholipase A2 hydrolyzes and cleaves an sn-2 thio ester. The fact that hydrolysis releases a free thiol group has been utilized as the basis for a spectrophotometric assay shown below in Scheme A. See also, Lin Yu and Edward A. Dennis, Methods in Enzymology, Vol. 197, 65-75 (1991). Similarly, phospholipase A1 hydrolyzes and cleaves an sn-1 thio ester. This sn-1 cleavage has also been utilized as the basis for a spectrophotometric assay. See, Kucera et al., Journal of Biological Chemistry, Vol. 263, 1264-1269 (1988). Further, the enzymatic activity of hepatic lipase, which catalzyes the hydrolysis of phospholipids, has also been measured via assay. See Deckelbaum R. J., et al., Biochemistry, 31, 8544-8551, 8545 (1992). 
Phospholipase A2 cleaves the sn-2 oxy ester of phospholipids; it will also hydrolyze an sn-2 thio ester. As shown in Scheme B below, the liberated thiol is allowed to react with a thiol-sensitive reagent, and the formation is measured continuously by monitoring the increase in absorption associated with its production. 
DTNB is used to detect the free thiol group. This reagent is commercially available from Aldrich (Milwaukee, Wis.). DTNB is preferable because it is sufficiently soluble in buffer such that stock solutions are aqueous.
The ability of phospholipases to cleave the thio ester bonds of unnatural substrates has also been utilized to develop continuous spectrophotometric assays for phospholipase A2 (PLA2), phospholipase A1 (PLA1), phospholipase C (PLC), lysophospholipase and lipase. The detection methods available for phospholipase assays include titrametric, acidimetric, radiometric, nuclear magnetic resonance and others, including the thio assay.
The thio assay possesses many characteristics that recommend it as a general assay for phospholipases. The most important are that it is a continuous, spectrophotometric assay which is very convenient, it directly detects one of the products liberated upon hydrolysis, it is one of the more sensitive assays, and it is also suitable for detailed kinetic studies. The thio assay can be used for phospholipases A1 and A2 and with appropriate modification of the substrate would be applicable to other phospholipases. However, owing to the lack of commercial availability of thiophospholipid substrates and their complicated synthesis, the thio assay has not been used extensively.
Although commonly employed in phospholipase assays, natural phospholipid substrates bring disadvantages to an assay due to their long fatty acid chains. Phospholipids with long fatty acid chains solubilize into mixed micelles in the presence of detergents commonly used in an assay. Long-chain phospholipids also form vesicles and liposomes, which serve as a membrane model. Vesicle phospholipid packing and phase transition characteristics are a disadvantage since they both dramatically affect enzymatic activity. This presents a problem when comparing hydrolysis rates for different phospholipids. It is difficult to determine if an apparent preference of an enzyme for one phospholipid or another represents true specificity of the enzyme or is simply due to the different phospholipid packing of phase transition temperatures of the two substrates. These factors can also be a problem when performing assays containing surface active agents, such as inhibitors.
There are a number of synthetic phospholipid substrates available. A major issue to consider when selecting a phospholipase assay is the choice of an appropriate synthetic substrate from the large number of phospholipids available. A number of thiophospholipids have been synthesized as substrates, including stereospecific didecanoyl-thio-phosphatidylcholine (thioPC) and its analog, didecanoyl-thio-phosphatidylethanolamine (thioPE). Using these substrates, the free thiol which is formed on hydrolysis of the thio ester bond is detected by the thio assay upon addition of either 5,5xe2x80x2-dithiobis(2-nitrobenzoic acid) (DTNB) or 4,4xe2x80x2-dithiobispyridine (DTP) as a thiol coloring reagent.
The thiol coloring reagents, DTP and DTNB, shown below, are routinely used to detect free thiol groups in the thio assay. 
Both are commercially available. The choice of which thiol coloring reagent is more advantageous to use in the thio assay depends on pH, solubility, the effect on the extinction coefficient, and the effect on the enzyme. Another important consideration in choice of which thiol coloring reagent to use in an assay is whether or not either of the above thiol reagents affects the activity of an enzyme.
Thus, the thio assay is a method used to measure phospholipase activity in an assay. The thio assay is incompatible with free thiols or any other substance which would significantly reduce the diaryl disulfide bond in the thiol coloring reagent and, as a consequence, is generally limited to the measurement of pure phospholipases. However, the thio assay is convenient and reproducible when compared to other types of assays. In addition, the thio assay has a lower limit of detection of 1 nmol/min.
Recently, interest has grown in the study of assay strategies and methods for phospholipases including the thio assay. See for example Reynolds, et al., Assay Strategies and Methods for Phospholipases, Methods in Enzymology, Vol. 197, 3-23 (1991); See also, Lin Yu and Edward Dennis, Thio-Based Phospholipase Assay, Methods in Enzymology, Vol. 197, 65-75 (1991).
As mentioned above, thioPC and thioPE are effective substrates in the phospholipase assay. However, there are several limitations in the use of thioPC and thioPE as substrates. First, a lengthy synthesis is required for the preparation of these substrates. Secondly, thioPC requires additional portions of enzyme in order to obtain a detectable color. Further, thioPC hydrolyzes slowly and can thus cause low sensitivity within the assay. Finally, thioPE is increasingly unavailable due to its complicated synthesis method. Thus, there is a need for a better chromogenic substrate to replace thioPC and thioPE for use as a detection reagent in an assay.
The present invention, an unnatural substrate, is like thioPC substrates because it allows DTNB to be present throughout assay hydrolysis; and, thus, color is produced as the substrate is hydrolyzed. Therefore, only one incubation period is required compared to the two or more incubation periods required for thioPC, which further requires additional enzyme for color. Further, when the product of the reagent of the present invention reacts with a thio coloring agent, it does not cause an extraneous effect on the activity of the enzyme. Given the number of factors involved in choosing a thio coloring reagent for the thio assay, it is advantageous to minimize extraneous effect on the enzyme to the greatest extent possible. The invention is also an improvement over thioPE because its synthesis is much less complicated and should therefore be more generally available.
Thus the present invention relates to a novel chromogenic substrate which is a useful indicator compound in an analytical test system for the spectrophotometric detection of an enzyme in a liquid test sample. In particular, it relates to a better chromogenic substrate in the previously discussed phospholipase A1 assay which is useful for spectrophotometric identification of potent inhibitors for treatment of phospholipase A1 disorder. Thus, this invention relates to a new chromogenic substrate in the phospholipase assay, the process of preparing the substrate and the use of the substrate in a phospholipase assay to identify a potent inhibitor for a phospholipase related disorder.
Further, it is the object of the present invention to provide an assay which can be incorporated into the solid, porous matrix of an analytical test device as an indicator compound for the measurement of an enzyme incorporated therein or in a liquid test sample applied thereto.
Thus, in one aspect, the present invention is directed to a compound of formula I: 
wherein:
each of Q and M is independently C5-C19 alkyl, alkenyl or alkynyl;
Y is (CH2)2OH or CH2CH(OH)CH2OH;
or a non-interfering salt thereof.
The present invention is also directed to a compound of formula (I) in which each of Q and M is independently C5-Cl9 n-alkyl, n-alkenyl or n-alkynyl.
With reference to formula (I) above, the following are particular values:
each of M and Q is independently pentyl, hexyl, heptyl, nonyl, or tridecanyl; and
Y is (CH2)2OH.
A more particular compound of formula (I) is one in which:
Q is nonyl.
Another particular compound of formula (I) is one in which:
each of Q and M is nonyl; and
Y is (CH2)2OH. (This particular compound is ThioPEG,
or a salt thereof, as exemplified in Example 1)
As used above, and throughout the description of the invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
xe2x80x9cAlkenylxe2x80x9d means an aliphatic hydrocarbon group containing carbon-carbon double bonds.
xe2x80x9cAlkylxe2x80x9d means an aliphatic hydrocarbon group which is straight or branched.
xe2x80x9cAlkynylxe2x80x9d means an aliphatic hydrocarbon group containing carbon-carbon triple bonds.
xe2x80x9cA compound of the present inventionxe2x80x9d or an equivalent expression, is meant to embrace a compound of general formula (I) as hereinbefore described, which expression includes a salt, where the context so permits. Similarly, reference to an intermediate, whether or not it itself is claimed, is meant to embrace a salt, where the context so permits. For the sake of clarity, particular instances when the context so permits are sometimes indicated in the text, but these instances are purely illustrative and it is not intended to exclude other instances when the context so permits.
This invention provides, in addition, a process for preparing a compound of formula I (or a non-interfering salt thereof) comprising:
(i) for a compound of formula I in which Y is (CH2)2OH, phosphorylating the hydroxy group of a corresponding compound of formula II: 
xe2x80x83using 2-chloro phospholane-2-oxide followed by treatment with water; or
(ii) phosphorylating the hydroxy group of an alcohol of forumula Y-OH using a compound of formula III: 
xe2x80x83followed by hydrolysis;
whereafter when a non-interfering salt is required such salt may be made with a base which affords a non-interfering cation;
wherein, unless otherwise specified, each of Q, Y and M has values as defined above.
Another particular compound is the compound of formula I which is (1-decanoylthio-1-deoxy-2-decanoyl-sn-glycero-3-phosphoryl)ethylene glycol.
Another particular compound is the compound of formula I which is 1-decanoylthio-1-deoxy-2-decanoyl-sn-glycero-3-phosphoryl-1xe2x80x2-sn-glycerol.
An additional aspect of the present invention is directed to a compound of formula III: 
wherein each of the values of Q and M is as defined above.
In the reactions described herein it may be necessary to protect reactive functional groups, for example hydroxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions. Conventional protecting groups may be used in accordance with standard practice, for examples see T. W. Green and P. G. M. Wuts in xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d John Wiley and Sons, Inc., 1999.
Thus, there is provided a process for preparing a novel compound of formula I as provided in any of the above descriptions which is selected from any of those described in the examples. This includes phosphorylation of the deprotected hydroxy group of a compound of formula II using commonly known phosporylating agents. The preferred phosphorylating agent, when Y is (CH2)2OH, is 2-chloro-phospholane-2-oxide. The preferred temperature for this reaction is from about 10xc2x0 C. to about 50xc2x0 C. The most preferred temperature for this reaction is about ambient temperature.
The present invention further provides a method of using the compound of formula I as an indicator in an analytical test system for the measurement of enzymatic activity in a liquid test sample.
The invention further provides for a method of using the compound of formula I as an indicator compound in analytical test systems for the measurement of enzymatic activity in a liquid test sample for the treatment of diseases.
The invention also provides for a method of using the compound of formula I in a phospholipase Al or B assay to spectrophotometrically identify an inhibitor for the treatment of a phospholipase A1 related disorder.
The invention further provides for a method of using the compound of formula I in the phospholipase A1 assay to identify a hepatic lipase inhibitor.
Thus, the present invention relates to a novel chromogenic substrate for use in an assay to afford identification of enzymatic activity in a screen.
The present invention further relates to a chromogenic substrate which may be used in an assay and will allow identification of a potent inhibitor such as a hepatic lipase inhibitor.
The present invention also relates to a chromogenic substrate which may be used in a phospholipase A1 assay and which will allow the identification of a potent inhibitor for the treatment of a phospholipase A1 related disorder(s).
The present invention also relates to a chromogenic substrate which may be used in a phospholipase A1 assay and which will allow the identification of a potent inhibitor for the treatment of a phospholipase A1 related disorder such as a hepatic lipase inhibitor to be used as a cardiovascular protective agent for raising plasma high-density lipoprotein (HDL) levels. In particular, this compound is a chromogenic substrate in the hepatic lipase inhibition assay in the presence of DTNB (Ellman""s Reagent), replacing thioPC.
Another special embodiment of the method of the present invention is the use of a compound of formula I in a phospholipase A1 assay to spectrophotometrically identify an inhibitor for the treatment of a phospholipase A1 related disorder.
Another special embodiment of the method of the present invention is the use of a substrate of formula I in a phospholipase A1 assay to identify a hepatic lipase inhibitor.
It is a further object of the invention to provide a kit or test device which may be effectively utilized for carrying out the novel uses of the invention.
In another aspect, a kit or test device is provided and comprises a compound of forumula I in a suitable container. Depending upon the purpose of the kit, a kit according to the invention can further include an additional or additional containers that contain, for example, a control reagent, a coloring agent and other reagents suitable for running an assay of interest.
In another aspect, the kit or test device for measurement of enzymatic activity contains a carrier matrix incorporated with the compound of formula I.
Thus, a novel chromogenic substrate is provided which has valuable properties. The substrate may be particularly useful in an assay for identification of an inhibitor of an enzyme. The inhibitor, in turn, may be useful for treatment of a phospholipase A1 related disorder. The substrate could be used in an assay to identify a hepatic lipase inhibitor, for example, which could then be used as cardiovascular protective agents for raising plasma high-density lipoprotein (HDL) levels.
In general, a compound of formula I may be prepared according to the route outlined in Scheme B below, and described in the Examples, in which each of M and Q, respectively, represents a value defined for the groups M and Q.
In Scheme C below, the hydroxy group of the epoxide of formula 1 is protected with a suitable protecting group such as trityl to form a protected epoxide of formula 2. The epoxide, formula 2, is ring opened with a thio fatty acid nucleophile to form the compound of formula 3. A compound of formula 3 is combined with sodium methoxide in methanol (methanolysis) yielding the compound of formula 4.
For a compound in which M is the same as Q, a compound of formula 5 is obtained via diacylation when compound 4 is treated with an activated derivative of an acid of formula QCOOH, for example, using the acid chloride in the presence of a base such as pyridine and a solvent such as methylene chloride, as described in Example 1. The compound of formula 5 is then deprotected to form a compound of formula II. The compound of formula II is phosphorylated as described in the Examples to form compound I. 
Utilizing an alternative procedure, Scheme D, values of M and Q may be different. In the alternative procedure, the epoxide of formula 2 is ring opened with a thio fatty acid nucleophile of formula MCOSxe2x88x92 where M is as previously defined, to form compound 8. An example of a thio fatty acid nucleophile is thio decanoyl acid sodium salt. The compounds of formula 5, II, and I are formed using methods similar to those described above in Scheme B and in the Examples.
Alternatively, as shown in Scheme D below, compound II may be phosphorylated with POCl3 to form a compound of formula III. The compound of formula III may be utilized to prepare the compounds of formula I wherein Y is CH2CH2OH (compound 9) or CH2CH2OHCH2OH (compound 10). To prepare compound 9 from a compound of formula III, ethylene glycol is treated with the compound of formula III, followed by an acid hydrolysis wash. Compound 10 may similarly be formed via reaction of a compound of formula III with glycerol followed by acid hydrolysis wash. 
If not commercially available, the necessary starting materials for the preparation of a compound of formula I may be prepared by procedures which are selected from standard techniques of organic chemistry, from techniques which are analogous to the syntheses of known, structurally similar compounds, and techniques which are analogous to the above described procedures or procedures described in the Examples. It will be clear to one skilled in the art that a variety of sequences is available for the preparation of the starting materials. Starting materials which are novel provide another aspect of the invention.
As mentioned above, the invention includes acceptable salts of the thioPEG substrate compounds defined by the above formula I. Acceptable salts are those that do not interfere with analytical methods and procedures. Such salt may be made with a base which affords an acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts, aluminum salts and ammonium salts, as well as salts made from organic bases such as triethylamine, morpholine, piperidine and triethanolamine. The potassium and sodium salt forms are particularly preferred.
Selective methods of protection and deprotection are well known in the art for preparation of compounds such as those corresponding to a compound of formula I.